PROJECT SUMMARY. An increase in muscle mass (hypertrophy) is a protective factor against many diseases whereas muscle atrophy worsens the quality of life and disease prognosis. Typically, muscle atrophy results from increased protein degradation, which is carried out primarily by the proteasome in skeletal muscle. This proteolytic system relies on ubiquitin ligases that ubiquitinate target proteins and determine their subsequent degradation. However, apart from a few well-known ubiquitin ligases that promote muscle atrophy, little is known about the role that the >600 ubiquitin ligases play in muscle homeostasis and about their specificity for protein ubiquitination. Moreover, it is largely unknown whether ubiquitin ligases that play a role in muscle atropy and hypertrophy also regulate sarcopenia, i.e. the loss of muscle mass and strength that occurs with aging. The ubiquitin ligases of the N-end rule pathway (UBRs) have been proposed to conjugate ubiquitin to proteins bearing N-terminal degradation signals that are derived from N-terminal cleavage or post-translational modifications. However, apart from a few target proteins, there is no comprehensive assessment of the proteins ubiquitinated by any UBR. Moreover, although implied by in vitro experiments with muscle extracts and pharmacologic inhibitors, the functional involvement of UBRs in myofiber size regulation remains unexplored. We have used developmental muscle growth in Drosophila as a convenient system for high-throughput screening and found several evolutionarily conserved ubiquitin ligases that regulate myofiber size, including the N-end rule ubiquitin ligase UBR4. UBR4 RNAi increases myofiber size in Drosophila and in mouse C2C12 myotubes. These findings provide evidence for a role of UBR4 in myofiber size regulation. On this premise, we propose to use biochemical and genetic approaches in mice and Drosophila to determine the following: (Aim 1) the role of UBR4 in muscle hypertrophy, starvation-induced atrophy, and sarcopenia in mice; (Aim 2) the UBR4 ubiquitination targets that are key for myofiber size regulation; and (Aim 3) the outcome of short- and long-term modulation of UBR4 activity on protein quality control and muscle strength in Drosophila and mice during aging. Altogether, these aims will provide a mechanistic understanding of the function of the N-end rule ubiquitin ligase UBR4 in muscle hypertrophy and sarcopenia and determine its primary ubiquitination targets. !